The aim of this study is to investigate in depth diabetes mellitus associated with immune checkpoint inhibitors (DM-ICIs) by analysing a case series. We also evaluated the clinical impact of flash glucose monitoring (FGM) systems in the management of this entity.  We conducted an observational cohort study of DM-ICIs diagnosed in two hospitals in Seville (Spain). Patients with a new diagnosis of diabetes mellitus (DM) or with sudden worsening of preexisting DM after starting treatment with ICIs, with a random 5 hour-postprandial C-peptide value of <0.6 nmol/L and without possibility of subsequent withdrawal of insulin treatment, were included. n = 6; 85.7%), with a mean age of 64.9 years. The mean glycated hemoglobin (HbA1c) upon diagnosis was 8.1%, with diabetic ketoacidosis (DKA) observed in 6 cases (85.7%). Subcutaneous flash glucose monitoring (FGM) systems were used in six cases, with a mean follow-up period of 42.7 weeks. During the first 90 days of use, mean average glucose was 167.5 mg/dL, with a coefficient of variation (CV) of 34.6%. The mean time in the range 70-180 mg/dL (TIR) was 59.7%, with a mean time above range (TAR) 181-250 mg/dL of 27.8% and a mean TAR > 250 mg/dL of 10.2%. The mean time below range (TBR) 54-69 mg/dL was 2%, while the mean TBR < 54 mg/dL was 0.3%. The mean glucose management indicator (GMI) was 7.3%. No significant differences were observed in FGM values for the following 90 days of follow-up. A progressive improvement in all parameters of glycaemic control was observed between the first month of FGM use and the sixth month of FGM use. Of note, there was a decrease in mean CV , mean TAR 181-250 , mean TAR > 250 mg/dL , mean TBR 54-69 mg/dL , and mean TBR < 54 mg/dL , along with an increase in mean values of TIR 70-180 mg/dL . The lack of statistical significance in the differences observed in the mean FGM values over the follow-up period may be related to the small sample size.  A total of 7 cases were identified, mostly males ( DM-ICI is recognised by a state of sudden-onset insulinopenia, often associated with DKA. The use of FGM systems may be a valid option for the effective management of DM-ICIs and for the prevention of severe hyperglycaemic and hypoglycaemic episodes in this condition. In the last few years, immune checkpoint inhibitors (ICIs) have positioned themselves as one of the therapeutic options with the greatest benefits in the treatment of different types of cancer. Their favourable effects have been proven in a wide variety of clinical scenarios, including an increase in survival in advanced metastatic tumours such as advanced melanoma , 2. ConsFrom a biomolecular point of view, ICIs act by blocking inhibitory pathways in T lymphocytes (T cells) mediated by CTLA-4 (cytotoxic T-lymphocyte antigen 4), PD-1 (programmed cell death protein-1), and PD-L1 (programmed death ligand 1), with the aim of avoiding immune tolerance and increasing the T-cell ability to identify and respond to tumour cells . As a coβ-cells, as a consequence of overactivation of T cells (mostly CD8+) in the context of treatment with ICIs [Diabetes mellitus associated with immune checkpoint inhibitors (DM-ICIs) is a rare condition in clinical practice. Its incidence is estimated to be around 1-2% among patients receiving treatment with ICIs, although an upward trend has been observed in recent years in relation to the increased presence of these drugs in cancer treatment regimens , 7. Frommostly CD+ in the mostly CD+ in the ith ICIs . Interesith ICIs , 11.Currently, knowledge about DM-ICIs is scarce, given the low number of available cases due to the exceptional nature of its onset. The identification of risk factors for its development or its association with a favourable tumour response has been the subject of recent research , 13. HowIn addition, the potential severity associated with this condition and the expected increase in its incidence as a consequence of the increasing indications for ICIs in cancer treatment regimens raise the need for the development of specific strategies for prevention and management of DM-ICIs .In this sense, the implementation of new technological tools such as flash glucose monitoring (FGM) tools could have a positive impact on patients with DM-ICIs, especially in the prevention of hypoglycaemia and severe hyperglycaemic crisis. In the International Time in Range Consensus, Battelino et al. have proposed glycaemic control targets based on metrics from interstitial glucose monitoring systems, such as a glycaemic variability target (as defined by a coefficient of variation (CV) ≤ 36%) or different percentages of time in glycaemic ranges, for 14-day periods of use with collection of more than 70% of the data . HoweverIn this paper, we report a series of seven cases of DM-ICIs diagnosed in two hospitals located in Spain, with special emphasis on the benefits of the use of FGM systems for the management of this condition.We conducted a retrospective observational study of DM-ICI cases referred during the 2018-2021 period to the Endocrinology and Nutrition Departments of University Hospital Virgen Macarena and University Hospital Virgen del Rocío in Seville (Spain).β-cell destruction as evidenced by a result < 0.6 nmol/L in a random 5 h postprandial C-peptide measurement (as it has been suggested by the ADA-EASD consensus report for the management of T1DM) [The following criteria were used to diagnose cases of DM-ICIs: (1) diagnosis of diabetes after receiving the first course of treatment with ICIs, (2) onset with an episode of acute hyperglycaemia ≥ 200 mg/dL or glycated hemoglobin (HbA1c) ≥ 6.5% , (3) demonstration of of T1DM) , 16.Patients with a history of type 2 diabetes (T2DM) or prediabetes who had an episode of acute deterioration of glucose control after starting treatment with ICIs, with evidence of newly developed insulinopenia associated with the need for insulin therapy, were also included .The state of glucose control prior to the development of DM-ICIs was assessed by baseline glycaemia levels recorded in routine monthly blood tests requested by the Oncology Center, together with HbA1c levels (determined by high-performance liquid chromatographic method) in case these where available. After diagnosis of DM-ICIs, islet autoantibodies (anti-GAD65 and anti-IA-2 autoantibodies) were measured by electrochemiluminescence immunoassay . Where available, human leukocyte antigen- (HLA-) II-DQ genotyping was determined by polymerase chain reaction and sequence specific oligonucleotide probe (PCR-SSOP), using the INNO-LIPA HLA-DQ model .Initially, follow-up of glycaemic control after diagnosis of DM-ICIs was performed by self-monitoring of blood glucose (SMBG) through glucometers in all cases. Subsequently, subcutaneous flash glucose monitoring (FGM) systems  were implemented in those cases in which it was technically possible, analysing glucose data from 30-day and 90-day ambulatory glucose profile (AGP) reports.p value < 0.05, considering p values < 0.1 as close to statistical significance. The statistical analysis software SPSS v.26 (IBM Statistics) was used to perform the statistical analysis.Quantitative data were expressed as mean, standard deviation (SD), median, and range (minimum-maximum). For the assessment of the evolution of AGP data, a repeated measures general linear model with Bonferroni correction was developed. The significance level was defined by a This study was conducted in accordance with the ethical standards proposed in the Declaration of Helsinki (1964) and in its subsequent modifications. Data were collected retrospectively as part of routine patient care, in accordance with the guidelines of the hospitals' Ethics Committees.A total of seven cases were identified. The overall characteristics of the study cohort are summarised in n = 6), and the overall mean age was 64.9 years . The mean body mass index was 26.11 kg/m2 . The cancer diagnosis was melanoma in 42.9% of cases , lung adenocarcinoma in 28.55% (n = 2), and urothelial carcinoma in 28.55% (n = 2). One case (14.3%) received treatment with Ipilimumab (anti-CTLA-4)/nivolumab (anti-PD1), while five cases (71.4%) received monotherapy with nivolumab, pembrolizumab, cetrelimab (anti-PD1 drugs), and one case (14.3%) with atezolizumab (anti-PD-L1). A personal history of T2DM was recorded in one case (14.3%) and prediabetes in another case (14.3%), while the remaining patients  had no history of diabetes.85.7% of the cases were male . The mean number of ICI treatment cycles administered prior to diagnosis was 4.3 .Out of the 7 cases, six had DKA at diagnosis, while one had isolated hyperglycaemia . Mean glBlood tests performed prior to the diagnosis of DM-ICIs as part of the usual cancer follow-up revealed an abnormal increase in fasting plasma glucose (between 106 and 148 mg/dL) in 4 out of 7 cases with respect to what these patients had shown in previous follow-up visits Figures . In the Mean HbA1c value at debut was 8.1% (65 mmol/mol), measured at a mean of 20 days after diagnosis of DM-ICIs. The C-peptide level was below 0.6 nmol/L in all cases, with a mean of 43.5 days comprised between DM-ICIs diagnosis and C-peptide measurement. C-peptide measurement was a random measurement performed within 5 hours postmeal, coinciding with blood glucose values between 100 and 200 mg/dL in all cases. In this regard, case 7 initially showed a detectable C-peptide level  which subsequently decreased to insulinopenic levels (0.03 nmol/L at 28 weeks after DM-ICIs diagnosis) .All cases required subcutaneous basal-bolus insulin regimen for the management of DM-ICIs. The overall mean insulin requirement amounted to 0.6 IU/Kg body weight/24 hours (SD: 0.13), remaining constant throughout the follow-up period, with no decrease observed during the first weeks after the onset of DM-ICIs . In two n = 2 anti-GAD65+, n = 3 anti-IA2+), while the other two cases (28.6%) were islet autoantibody-negative. The HLA-II DQ genotype was explored in n = 3 cases, with two of them showing the DQA1∗05 : 01/DQB1∗02 : 01 haplotype  showed single islet autoantibody positivity  .In 6 out of 7 cases patients 1-6; 85.7%), FGM systems were used as part of the glycaemic control optimisation strategy, activating audible and vibrating alarms for hypoglycaemia (<70 mg/dL) and hyperglycaemia (>250 mg/dL). Initiation of use of these devices occurred after a mean of 28.7 weeks from diagnosis of DM-ICIs SD: 35.7 weeks) and continued for a mean of 42.7 weeks thereafter (SD: 20.3 weeks) , along with TAR > 250 mg/dL .Analysing the FGM data through the monthly AGP reports, we observed a progressive improvement in all parameters of glycaemic control from the first month of use of the FGM devices . Thus, Cp value = 0.09) as well as a decrease in TBR 54-69 mg/dL  and TBR < 54 mg/dL   .The percentage of time FGM sensor was active was higher than 80% in all cases at all timepoints, thus supporting a correct interpretation of FGM data in our cohort. Indeed, according to the International Consensus on Time in Range , the perFive patients (71.4%) discontinued ICI treatment after diagnosis of DM-ICIs, while 2 (28.6%) patients continued the scheduled treatment regimen. Of the five patients who discontinued treatment, one did so permanently. Four patients resumed ICI treatment after a mean of 14 weeks , and no deterioration in glucose control was observed in any of the cases (who remained on insulin therapy).Once the diagnosis was made, coordinated clinical visits were scheduled between Oncology and Endocrinology Centers for close clinical follow-up. The possibility of an unscheduled clinical assessment at the Diabetes Day Hospital of the Endocrinology Department was made available in the case there were alterations in glycaemic control requiring preferential or urgent clinical assessment.n = 2; n = 3 cases (42.8%), stable disease in n = 2 cases (28.6%), partial response in n = 1 case (14.3%), and disease progression (with exitus) in n = 1 case  had DKA at diagnosis.DM-ICIs is characterised by a state of insulinopenia with a sudden onset, leading to a high percentage of cases developing DKA at the time of clinical presentation (more than 67%) , 21. In Based on this mechanism, a C-peptide measurement below 0.6 nmol/L has been proposed as a diagnostic criterion for DM-ICIs by most authors , 12–14. β-cell destruction in DM-ICIs. In addition, some studies have suggested that DM-ICIs may be characterised by dysregulations of pancreatic α-cells and incretin system, with implications for α-cell glucagon production and/or incretin secretion  [Data from the FGM devices show that suboptimal control with a tendency to hyperglycemic episodes was maintained throughout the follow-up period, without a decrease in daily insulin requirements, which supports the theory of massive and persistent pancreatic cretion) , 25. Thicretion) , 26.In DM-ICIs, HbA1c levels are often not markedly high as compared to the observed glucose values that sometimes are extremely high at the time of diagnosis . This fiThe development of DM-ICIs in patients with a previous history of T2DM constitutes an entity with particular characteristics, with some cases reported in the current literature . HoweverIn our cohort, in one case (patient 7), C-peptide measurement during the first 24 hours after DM-ICIs diagnosis showed a normal value (0.9 nmol/L), which fell into the insulinopenic range in a subsequent measurement. In this regard, some authors propose a second confirmatory C-peptide measurement at least 15 days after DM-ICI diagnosis, or measurement under glucagon stimulation or with a mixed meal tolerance test in case of doubt . The decn = 5) were positive for islet autoantibodies, a percentage that is slightly higher than that reported in the main reports on DM-ICIs, which place it at around 50% [In our research, 71.4% of cases  within the genetic risk assessment for the development of autoimmune diabetes [∗03 binding imbalances  and, like other HLA class II polymorphisms such as DR4-DQ8 (DRB1∗04-DQA1∗03-DQB1∗03 : 02), has been associated with a strong risk for T1DM development [In the present case series, two cases (out of three studied) had the haplotype DQA1diabetes . This haelopment , 18, 35.elopment , 36. Howelopment . This poelopment , 20, 37.α cells), together with impaired exocrine pancreatic function [Some authors have suggested the existence of different subtypes of DM-ICIs depending on the clinical and biochemical profile at the onset . Thus, tfunction , 26. In function , 25. Thefunction .In line with this insight, in our series, 4 cases (57.1%) showed features compatible with a course similar to that of AFD, while 2 cases showed mixed features (negative islet autoantibodies) and 1 case showed a slower development time, with an onset in the form of isolated hyperglycaemia without DKA. Interestingly, the AGP reports extracted from the FGM devices showed how the patient diagnosed following an episode of isolated hyperglycaemia (patient 4) had a better glucose control compared to the other patients during postonset follow-up, with the highest TIR 70-180 mg/dL and the lowest CV (as compared to the other cases) and with a TBR < 70 mg/dL of less than 1.5%. Assuming the limitations derived from the analysis of a single case, this fact would support the theory of the existence of different onset forms of DM-ICIs, including one that shares clinical features comparable to those of typical T1DM.Different strategies have been proposed for the early identification of DM-ICIs. Some of them are providing health education notions focused on the identification of warning symptoms or the design of care circuits for specific assessment. In those cases, in the presence of a history of established T2DM or prediabetes, it is recommended to optimise glucose control –11, 19. Periodic blood tests performed at the same time of the treatment cycle administration may be useful to identify discrete variations in glycaemia levels that precede the debut of DM-ICIs . In our On the other hand, Fujiwara et al. have described changes in insulin secretion dynamics after initiation of treatment with ICIs in patients who subsequently developed DM-ICIs , so thatIn the event of a hyperglycaemic crisis in a patient being treated with ICIs, a differential diagnosis with similar conditions should be considered . The preIn terms of initial therapeutic management, the use of corticosteroids or other immunomodulators has not been shown to be effective in the treatment of DM-ICIs due to the frequent irreversibility of insulin secretion dysfunction, unlike other nonendocrine irAEs , 42. A cOn the other hand, given the difficulty of managing this condition, close and coordinated follow-up with other specialties could have added advantages in optimising the metabolic control of patients, allowing complications to be resolved at an early stage and progressively increasing the degree of autonomy in the management of diabetes. In this sense, multidisciplinary collaboration between different specialties (including oncologists and endocrinologists) plays a key role .p value > 0.05), probably due to the low number of cases in our cohort as a consequence of the rarity of this diabetes type. Yet, the increase in TIR and the decrease in TAR > 250 were close to statistical significance , suggesting that with a larger sample size, these differences could become statistically significant.The complexity of clinical management and adjustment of insulin therapy in DM-ICIs makes early implantation of continuous glucose monitoring systems a very useful tool in patients with this condition. In our case series, the benefits associated with FGM resulted in a progressive and generalised improvement in glucose parameters between the first and subsequent months of FGM use. In this regard, the CGM metrics observed decrease in CV and TBR < 70 mg/dL and TBR < 54 mg/dL was noteworthy, which is remarkable given the risk factors for the development of hypoglycaemia associated with this condition . We also observed a progressive increase in TIR 70-180 mg/dL, which overall was higher than 54% during the entire follow-up; also, we observed a decrease in TAR > 250 mg/dL from 16.3% in the first month to 7.7% in the sixth month. These differences failed to reach statistical significance  may limit the interpretation of our study conclusions, requiring comparison of our results with other similar cohorts. Second, the lack of measurement of C-peptide and islet autoantibodies at baseline limits the interpretation of the process through which DM-ICIs develop. The measurement of zinc transporter 8 autoantibodies  was not available in our setting, nor was the measurement of the HLA-DR haplotypes. HLA-DQ haplotypes could only be determined in three patients due to factors external to the investigation. On the other hand, use of FGM systems was relatively delayed after the disease onset; therefore, FGM use may not entirely reflect the exact patterns of blood glucose derangements in our cohort. In addition, longer-term follow-up of patients with DM-ICIs  would confirm the theory of a persistent insulin secretion dysfunction that cannot be reversed.In conclusion, DM-ICI is a condition characterised by a high complexity in terms of management, in which an early diagnosis allows the prompt initiation of insulin treatment and the use of technological systems (such as FGM devices) that represent strategies leading to significant clinical benefits, including improvement of glucose control, prevention of hypoglycemic episodes, and reduction of hospital readmissions. In this regard, the presence of an endocrinologist in the multidisciplinary team around the cancer patients is a crucial aspect since it facilitates the implementation of coordinated strategies during the follow-up.Therefore, large prospective studies using continuous glucose monitoring systems are needed to better investigate the patterns of glucose derangements in patients with DM-ICIs and to understand the most effective therapeutic interventions in this population.


Our study provides substantial evidence that hydrogen-rich water can prevent intestinal damage caused by LPS-induced stimulation. Intestinal injury caused by LPS is related to the NFκB signaling pathway. LPS promotes the generation of inflammatory factors and oxygen free radicals by activating the NFκB signaling pathway in intestinal cells, causing inflammation and oxidative stress and destroying the tight-junction state of the intestine, causing intestinal damage. Hydrogen-rich water can activate the Nrf-2 signaling pathway to remove excess oxygen free radicals and relieve oxidative stress. The Nrf-2 signaling pathway, an upstream signaling pathway of NFκB, can act as a protective mechanism against inflammation by regulating NFκB. In summary, hydrogen-rich water can inhibit oxidative stress and regulate NF by activating Nrf-2 signal pathway κB signal pathway, inhibit the production of inflammatory cytokines, maintain the tight connection in the intestine, and protect the intestine from damage caused by lipopolysaccharide. Our research contributes to further understanding of the mechanism of hydrogen-rich water, as well as its value for clinical application.Long-term exposure to low-dose lipopolysaccharide can impair intestinal barriers, causing intestinal inflammation and leading to systemic inflammation. Hydrogen-rich water possesses antioxidant and anti-inflammatory functions and exerts inhibitory effects on various inflammatory diseases. In this study, we investigated whether oral hydrogen-rich water could prevent lipopolysaccharide-induced chronic intestinal inflammation. An experimental model was established by feeding hydrogen-rich water, followed by the injection of lipopolysaccharide (200 μg/kg) in the tail vein of rats after seven months. ELISA, Western blot, immunohistochemistry, and other methods were used to detect related cytokines, proteins related to the NF-κB and Nrf-2 signaling pathways, and tight-junction proteins to study the anti-inflammatory and antioxidant effects of hydrogen-rich water. The obtained results show that hydrogen-rich water significantly increased the levels of superoxide dismutase and structural proteins; activated the Nrf-2 signaling pathway; downregulated the expression of inflammatory factors cyclooxygenase-2, myeloperoxidase, and ROS; and decreased the activation of the NF-κB signaling pathway. These results suggest that hydrogen-rich water could protect against chronic intestinal inflammation in rats caused by lipopolysaccharide-induced activation of the NF-κB signaling pathway by regulating the Nrf-2 signaling pathway. The Nrf-2 and NFκB signaling pathways are known to cause various diseases. Inflammatory responses are tightly regulated by pro- and anti-inflammatory mediators, critical factors affecting the development of inflammatory bowel disease (IBD), obesity, and neurological and systemic diseases . ChronicA significant part of food digestion occurs in the small intestine, which makes up a large portion of the body’s internal surface. The functional and morphological integrity of the small intestine is vital to prevent toxic substances from damaging the tight-junction barrier. The dysregulation of the gut barrier can result in several pathological conditions in the intestines, such as the accumulation of endotoxin, which could result in an inflammatory response in the intestine . Damage Tight-junction proteins are connected via multiprotein complexes. These tight-junction proteins are located in the apical portion of the lateral membrane of epithelial cells. During physiological activities, tight-junction proteins protect the solute from the surroundings via the intercellular space . Tight jLipopolysaccharides (LPS) (endotoxin) are the main component of the outer membrane of Gram-negative bacteria. They induce cellular apoptosis, increase epithelial cell permeability, and produce DNA damage. Prolonged exposure to LPS has been shown to activate the proinflammatory response mediated by the NF-κB pathway, thereby compromising the intestinal barrier . TherefoThere has been a growing interest in hydrogen (H2), owing to its numerous biological activities, such as antioxidant properties, as per the results of animal testing . Ohsawa HRW has exhibited various biological roles as a therapeutic antioxidant, such as anti-inflammatory and antioxidant effects. Therefore, the aim of this study was to explore whether oral administration of HRW could alleviate inflammatory responses in LPS-induced rat intestinal inflammation.Sprague–Dawley  rats weighing 220 ± 20 g  were procured from Baikui Tianxing Farm, Hulan District, Harbin City. The rats were raised in the Animal Breeding Center of Northeast Agricultural University under a controlled temperature of 20 ± 1 °C and at a relative humidity of 55–60% with a 12 h/12 h light/dark cycle. Animals had free access to water and were fed a standard diet. All rats were allowed to acclimatize for one week before starting the experiments. This study was approved by the Ethical Committee for Animal Experiments NEAUC2020 04 17 .Hydrogen generated by a hydrogen generator  was stored in a bottle and soaked in 400 mL of distilled water at a rate of 500 mL/min using the exhaust method. It took 15 min to reach saturation, resulting in hydrogen-rich .LPS injection  was diluted using sterile deionized water to a final concentration of 200 μg/kg before use .Rats were randomly assigned to four experimental groups . Drinking time was limited at specific time points . All rats had free access to food. A: The control group (CON) was fed with distilled water for seven months; after seven months, sterile normal saline (concentration: 0.5 mL/kg body weight) was injected via the tail vein on the first day of every week. B: The hydrogen-rich water group (HRW) was fed HRW for seven months; after seven months, sterile saline was injected into the tail vein on the first day of the week in the same concentration as that of the CON group. C: The tail vein injection of lipopolysaccharide (LPS) group was fed with distilled water for seven months; after seven months, LPS (concentration: 200 μg/kg body weight) was injected into the tail vein on the first day of every week. D: The HRW plus lipopolysaccharide injection through the tail vein (HRW + LPS) group was HRW for seven months; after one month, LPS was injected into the tail vein on the first day of every week in the same concentration as that of the LPS group. All rats underwent a tail vein blood draw every weekend for four weeks.g, 4), and serum was stored at −80 °C. The ileum was removed, and the intestinal contents were washed with PBS and divided into two parts; one part was fixed with 10% formalin for histological, immunofluorescence, and immunohistochemical analysis, and the other part was used for ELISA, RNA extraction, real-time quantification, and Western blotting.Weight changes in the rats were recorded weekly. After four weeks, one hour before sacrifice, the rats were administered ovalbumin (OVA) by gavage, the concentration of OVA was 250 mg·kg-1·bw-1 one hour after gavage. The permeability of the intestine to macromolecular substances was assessed by measuring OVA in serum . The rathttps://www.ncbi.nlm.nih.gov). After verification, the primers were synthesized by Sangon Bioengineering Company at a concentration of 10 μM. qRT-PCR was performed using a Roche Light Cycler 480 real-time PCR instrument , and each sample was run in 3 replicates. Data were collected, and the relative mRNA expression of target genes was assessed using the 2−ΔΔ Ct method. Total RNA was isolated from ileum tissues using Trizol reagent . The RNA concentration and purity of all samples were determined on a NanoDrop spectrophotometer . Reverse RNA transcription was performed using a reverse transcription kit . Sangon Bioengineering  Co., Ltd. designed the primers. Homology searches were performed using BLAST from the NCBI , myeloperoxidase (MPO), ovalbumin (OVA), malondialdehyde (MDA), and superoxide dismutase (SOD) in the ileum tissue were determined using an ELISA kit . The absorbance  was measured at a wavelength of 450 nm using an Epoch microplate reader , and the sample concentration was calculated.The total protein was extracted from ileum tissue samples using RIPA lysis buffer . The protein concentration was determined using a BCA protein assay kit . SDS-PAGE gels were prepared on glass plates using an SDS-PAGE gel kit . A volume of 5 μL of protein samples was loaded per well for SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% nonfat dry milk  in 1 × TBST for 120 min and incubated in corresponding primary antibodies , then incubated overnight at 4 °C. We analyzed the effects of HRW and LPS on the NF-κB and Nrf-2 signaling pathways by detecting changes in these proteins. The next day, samples were washed three times with one × TBST and incubated with horseradish peroxidase (HRP)-labeled secondary antibody  on the membrane for two h on a shaker at room temperature. Then, the samples were washed thrice with one × TBST again, followed by color development using ECL chemiluminescence . Images were collected using a Tanon-5200 chemiluminescent imaging system . The protein bands were analyzed and quantified using ImageJ software .The ileum pathologically analyzed to observe the intestinal structure, the arrangement of the ileal villi, the presence of lesions, and the infiltration of inflammatory cells to evaluate the protective effect of hydrogen-rich water on the intestinal injury. After the rats were sacrificed, intestinal tissues were preserved in a 10% formalin fixative solution, removed, placed in cassettes for slicing, and rinsed overnight in running water. The samples were dehydrated through gradient alcohol and cleared using xylene. Next, the samples were immersed in wax, embedded in paraffin, and cut into five μm thick sections. These sections were stained with hematoxylin and eosin for histopathological analysis. The samples were sliced in a crosscut ring direction.2O2 for 15 min. The slides were blocked with 3% BSA. Then, samples were diluted 1:100 with primary anti-IgG antibody  overnight. Subsequently, sections were washed with wash buffer, followed by coincubation with 3,3-O-diaminobenzidine (DAB) solution and horseradish peroxidase (HRP) in the dark. Each sample was left at room temperature for 30 min. Sections were counterstained with hematoxylin, dehydrated using xylene, and mounted in DPX. Slides were observed under a light microscope. The images were acquired at 150× magnification; yellow or brown–yellow indicated positive staining, with three dyes per group, and stain intensity was quantitated using ImageJ software .The levels of ZO-1, occludin, and claudin-1 were detected by immunohistochemistry, and the infiltration of macrophages was determined by detecting CD68. Immunohistochemistry was performed on five μm thick sections after deparaffinization following the established protocol. The sections were dehydrated in conventional gradient alcohol, cleared with xylene, and washed with PBS. The sections were incubated in proteinase K (20 μg/mL) for 20 min at room temperature, followed by rinsing with TBS. Endogenous peroxidase activity was blocked by incubating the slides with 3% HROS content was detected in rat ileum by immunofluorescence. The slides were dewaxed in xylene and rehydrated using graded ethanol. Next, the sections were incubated with anti-ROS antibody (1:1000)  for 1 h at room temperature, followed by 30 min incubation with a secondary antibody and a brief wash. Slices were observed using a fluorescence microscope.t-test or one-way ANOVA test was performed to determine significant differences; *, # p < 0.05; **, ## p < 0.01.Results were analyzed and graphed using Prism 7.0 software . The results were expressed as mean ± standard deviation (mean ± SD). A Student’s Rat gut structure was histologically analyzed to investigate the integrity of the gut barrier. The ileum structure of the CON group was complete and transparent A. The vip < 0.01). Compared with the LPS group, the expressions of ZO-1, occludin, and claudin-1 increased in the HRW + LPS group (p < 0.01). Additionally, the OVA content was significantly increased in the LPS group compared with the CON group (p < 0.01). In contrast, the OVA content was significantly decreased in the HRW + LPS group (p < 0.01).The integrity of the intestinal barrier structure forms the basis of intestinal function. Here, the effect of HRW on gut integrity was examined by measuring OVA concentrations and tight-junction protein expression. The expression of tight-junction proteins is a crucial indicator of intestinal mucosal integrity. The expression of tight-junction proteins in ileal tissue was determined. Compared with the CON group, the expressions of ZO-1, occludin, and claudin-1 were significantly reduced in the intestinal tissue of the LPS group (p < 0.01). However, the concentrations of IL-6 and TNF-α in the HRW + LPS group were decreased in the serum compared with the LPS group (p < 0.05). Additionally, the concentrations of IL-1β and MCP-1 were significantly reduced (p < 0.01). In the intestinal tissue, compared with the LPS group, the HRW + LPS group had decreased the levels of IL-1β and TNF-α (p < 0.05), and the expression of IL-6 and MCP-1 was decreased significantly (p < 0.01). qPCR data showed that the relative gene expression of inflammatory cytokines in rat ileal tissue was significantly upregulated after LPS treatment (p < 0.01). The expression of cytokine-related genes in the tract tissue was significantly inhibited, and the relative gene expression of cytokines in the HRW + LPS group was significantly downregulated compared with the LPS group (p < 0.01). In this study, we determined the content of COX-2 in rat ileal tissue, which responded to inflammatory responses in rat intestinal tissue. Compared with the CON group, the COX-2 content in the tissues of the LPS group was significantly enhanced (p < 0.01). However, the COX-2 content in the tissues of the HRW + LPS group was significantly decreased compared with the LPS group (p < 0.01).Compared with the CON group, IL-1β, IL-6, TNF-α, and MCP-1 levels in serum and tissues were significantly elevated in the LPS group (p < 0.01). In contrast, the HRW + LPS group had a significantly decreased number of macrophages in ileal tissue compared with the LPS group (p < 0.01).Compared with the CON group, the number of macrophages in the intestinal tissue was significantly increased in the LPS group (p < 0.01). In contrast, the protein expressions of TLR4 and MyD88 in the HRW + LPS group were lower compared with those in the LPS group (p < 0.05). The expression of IκBα protein was significantly reduced in the LPS group (p < 0.01), but it was increased in the HRW + LPS group (p < 0.05). HRW pretreatment upregulated the expression of IκBα protein (p < 0.05), and p-IκBα protein expression was significantly increased in the HRW + LPS group compared with LPS group (p < 0.01). The expressions of p-p50 and p-p65 in the LPS group were significantly increased (p < 0.01), whereas the expression of p-p50 and p-p65 in HRW + LPS group was inhibited (p < 0.05).In this study, the expression of NF-κB signaling pathway-related proteins was measured in each group A. Comparp < 0.01). Compared with the LPS group, the HRW + LPS group had significantly reduced ROS levels (p < 0.01). Furthermore, MPO levels were measured in rat intestinal tissue. Compared with the CON group, the MPO content in the LPS group was significantly increased (p < 0.01), and compared with the LPS group, the MPO content was significantly decreased in the HRW + LPS group (p < 0.01).ROS content was determined in intestinal tissue by immunofluorescence technique A. Comparp < 0.05), whereas the SOD content in the HRW group was significantly increased (p < 0.01). Compared with the LPS group, the SOD content in the HRW + LPS group was significantly increased (p < 0.01). Compared with the CON group, the MDA content in the LPS group was significantly enhanced (p < 0.01). Compared with the LPS group, the MDA content in the HRW + LPS group was significantly decreased (p < 0.01).Compared with the CON group, the SOD content in the tissues in the LPS group was decreased (p < 0.01), and the expressions of HO-1 and NQO1 protein decreased in the LPS group . Compared with the LPS group, Nrf-2, HO-1, and NQO1 protein levels were significantly increased in the HRW + LPS group . The expression of Nrf-2 signaling pathway-related proteins was measured in each group . CompareIn recent years, hydrogen molecules have been shown to exhibit antioxidative and anti-inflammatory effects in research on human and animals , allowinPrevious studies have shown that many inflammatory processes induced by LPS cause oxidative damage . ImbalanOxidative stress and inflammation are interrelated in various pathological events. Previous studies have shown that the production of inflammatory cytokines is associated with the activation of NF-κB ,45,46, wLPS stimulation is known to activate the NF-κB signaling pathway, leading to an influx of chemotactic cytokines and peripheral circulating macrophages into the intestinal epithelial barrier . In addiTight junctions consist of more than 30 structural or functional proteins . They haAdditionally, severe disruption of ileal histology was observed following H&E staining, which showed that LPS-induced inflammation resulted in the impairment of small intestinal barrier integrity, consistent with the results of previous studies ,71. HoweIn the present study, activation of the NF-κB signaling pathway by LPS resulted in intestinal and systemic inflammation accompanied by increasing intestinal oxidative stress and intestinal permeability. HRW increased the expression of tight-junction proteins, improving the antioxidant capacity of rats, reducing the content of ROS and MPO, alleviating the level of macrophage infiltration, activating the expression of the Nrf-2 signaling pathway, and regulating the NF-κB signaling pathway, thereby reducing LPS-induced intestinal chronic inflammation in rats, with positive antioxidant and anti-inflammatory effects.


Many studies have compared real-world clinical outcomes of immunotherapy in patients with metastatic non-small cell lung cancer (NSCLC) with reported outcomes data from pivotal trials. However, any differences observed could be only limitedly explored further for causation because of the unavailability of individual patient data (IPD) from trial participants. The present study aims to explore the additional benefit of comparison with IPD.n = 141) with IPD from participants in the Checkmate-057 clinical trial (n = 292). Univariate and multivariate Cox proportional hazards models were used to construct HRs for real-world practice versus clinical trial.This study compares progression free survival (PFS) and overall survival (OS) of metastatic NSCLC patients treated with second line nivolumab in real-world clinical practice , had more often ECOG PS ≥ 2 (5 vs. 0%) and were less often treated with subsequent anti-cancer treatment (28.4 vs. 42.5%) compared to trial patients. The median PFS in real-world patients was longer (3.84 (95%CI: 3.19-5.49) vs 2.30 (2.20-3.50) months) and the OS shorter than in trial participants (8.25 (6.93-13.2) vs. 12.2 (9.90-15.1) months). Adjustment with available patient characteristics, led to a shift in the hazard ratio (HR) for OS, but not for PFS (HRs from 1.13 (0.88-1.44) to 1.07 (0.83-1.38), and from 0.82 (0.66-1.03) to 0.79 (0.63-1.00), respectively).This study is an example how IPD from both real-world and trial patients can be applied to search for factors that could explain an efficacy-effectiveness gap. Making IPD from clinical trials available to the international research community allows this.The online version contains supplementary material available at 10.1186/s12874-022-01760-0. The treatment landscape of metastatic lung cancer patients has changed over recent years . ChemothThe phase-III marketing authorization trials researching immunotherapy in NSCLC patients used strict in- and exclusion criteria , 6, 7. PRecently, for one of the pivotal trials involved in the Dutch EE gap study, the IPD have come available. The aim of the present study is to explore if individual patient data (IPD) could be helpful to identify factors that explain divergence between outcomes from the nivolumab treatment arm of the Checkmate-057 clinical trial and patients with NSCLC treated in real-world clinical practice.. The data from that study were re-used. The trial data from the Checkmate-057 trial were collected from the internal ICI database of the Medicines Evaluation Board database.This exploratory study is an in-depth study of the study of Cramer-van der Welle et al . The datThe population under study consisted of metastatic nonsquamous NSCLC patients treated with second line nivolumab after prior platinum-containing chemotherapy. Real-world patients were treated with nivolumab in the years 2015 to 2018. Participants in the Checkmate-057 clinical trial were treated before marketing authorization . The outPatient- and tumor characteristics of the study population were analyzed using descriptive statistics. These included age, gender, stage, Eastern Cooperative Oncology Group Performance Score (ECOG PS), the presence of brain metastases at diagnosis, tumor histology, and programmed death-ligand 1 (PD-L1) expression. Age was categorized in < 70 and ≥ 70 years, since NSCLC has a median onset at age 70 years .The Kaplan-Meier method with log-rank test was used to compare the PFS and OS between real-world and trial patients. Survival times were calculated from the start of nivolumab treatment  or randomization date . Patients not reaching the endpoint at data cut-off were censored at the last known alive date. Median follow-up duration was calculated for the study population using the reverse Kaplan-Meier method .p-value was < 0.05.Next, analogous to identification of potential confounders, relative changes in the HR were used to identify factors that could explain the difference between real-world and trial patient outcomes. To do so, univariable and multivariable Cox proportional hazards models were used to construct HRs for real-world practice versus clinical trial patients for both outcomes. All patient- and tumor characteristics (see above) were assessed as potential explanatory factors. Theoretically, variables that result in adjustment of the HR towards 1.00 were considered as potential causative for the EE-gap. Since this study does not compare two different treatments but two groups treated similarly, we argue that the influence of long-term survivors on the proportionality of the Cox model is limited. Statistical analyses were stated significant if the Data handling and statistical analyses were performed using the R software system for statistical computing  . For this secondary analysis, all clinical information was provided anonymously.p = 0.003) compared to trial patients. Five percent (n = 7) of the real-world patients had an ECOG PS of 2, compared to 0% in trial patients. The trial patients were more often treated with subsequent anti-cancer treatment compared to real-world patients . These characteristics are presented in Table A total of 292 metastatic NSCLC patients were treated with nivolumab in the Checkmate-057 trial and 141 patients in real-world clinical practice. The median follow-up time of the real-world and trial patients was respectively 25.2 (95%CI 22.7-32.6) and 18.6 (95%CI 17.6-20.1) months. Real-world patients were older (64 (44-80) years vs 61 (37-84), ECOG PS = Eastern Cooperative Oncology Group Performance Score, PD-L1 = Programmed death-ligand 1, RCT = randomized controlled trial.p = 0.104)  and PD-L1 expression > 50% (p < 0.001)  months compared to 2.30 (95%CI: 2.20-3.50) months in trial patients   and ECOG PS 2 (p = 0.001), and PD-L1 expression > 50% (p = 0.001) were significantly associated with OS (Table The median OS was 8.25 (95%CI: 6.93-13.2) months for real-world patients and 12.2 (95%CI: 9.90-15.1) months for trial patients  after adjustment, suggesting that differences in the available characteristics between the two settings partly explain the altered OS seen in real-world practice. The latter phenomenon was not observed for PFS, suggesting that for that outcome other unmeasured factors are involved.(supplement 1). Furthermore, measuring progressive disease using the Response Evaluation Criteria in Solid Tumors (RECIST)- criteria can be less structured and strict in real-world than in trial patients [The median PFS of real-world patients was longer compared to trial patients, resulting in an HR for PFS below 1.00. Although ECOG PS was statistically significant in the multivariate Cox analyses, the adjusted HR between real-world and trial patients did not change. The etiology for this gap in PFS is believed to be multifactorial, with contributing factors including differences in patient populations, healthcare delivery, and variability in the experience of treating health care providers. Multiple factors which could explain differences in patient populations were measured but did not lead to a difference in HR. Unmeasured factors involving PFS could be smoking status, comorbidities, and frailty. Previous research also showed that use of corticosteroids and the number of organs with metastases are associated with PFS . Healthcpatients . In clinpatients . ConsequIn contrast to PFS, the non-significant difference in OS between real-world and trial shifted towards a null effect after adjustment for the available characteristics in the data ). This suggests that differences in ECOG PS and presence of brain metastases are linked to the observed shorter OS in real-world practice.Apart from the beforementioned potential, this study also confirms the results using the standard approach of trial and real-world comparison using software applications. The unadjusted calculated HRs for PFS and OS in the study of Cramer-van der Welle et al are identical to the findings of this study using IPD .A strength of our study we consider the quality of the real-world data. Data were manually extracted from electronic healthcare records and with very few missing data. An exception is the PD-L1 expression status which was often missing in real-world 48.9%) since it is not mandatory to measure this before nivolumab treatment in second line. We therefore could not use this factor in the multivariate analyses. Besides this, we could also not test for smoking status that in the Checkmate-057 study was an effect modifier (less effect in never smokers). On the other hand, we expect most patients to be current or past smokers. Altogether, we argue that most of the characteristics with the high prognostic value were included in the analyses 8.9% sinc. This veClinicalStudyDataRequest.com will help to improve the availability of much more clinical trial data, considering the privacy of patients included in the trial, to allow better identification of factors associated with an efficacy-effectiveness gap (if any), in turn facilitating individualized prognoses and treatment planning [In the present study we assessed the value of IPD with second line nivolumab, while Cramer-van der Welle et al. also reported a significant impaired OS in real-world with first line pembrolizumab. Unfortunately, due to unavailability of trial IPD on pembrolizumab, we could not assess what the added value of adjustment with IPD would be for that regimen. The European Medicines Agency (EMA) started an initiative to publish clinical trial data submitted to EMA as part of marketing authorization applications . At the planning –23.This study is an example how IPD from both real-world and trial patients can be applied to search for factors that could explain an efficacy-effectiveness gap. Making IPD from clinical trials available to the international research community allows this.Additional file 1.


Fragility hip fracture (FHF) is a serious complication of osteoporosis. A fracture liaison service (FLS) is crucial in preventing FHF. Our retrospective data of 489 patients with FHF and 3-year follow-ups demonstrated that the FLS improved functional outcomes. Our study’s mortality rates were lower than in other published series.This study assessed the 3-year outcomes after fragility hip fracture (FHF) treatment by a multidisciplinary team from the Siriraj Fracture Liaison Service (Si–FLS). The review investigated the administration rates of anti-osteoporosis medication, refracture, and mortality; activities of daily living; mobility; and health-related quality of life.A retrospective review was performed of the records of Si-FLS patients given FHF treatment between June 2016 and October 2018. The outcomes were evaluated at 3 time points: before discharge, and 1 and 3 years after treatment.The study enrolled 489 patients . The mortality and refracture rates at 1 year after hip fracture were 13.9% and 1.6%, respectively. At the 3-year follow-up, both rates were higher . The Barthel Index and EuroQoL Visual Analogue Scale had risen to a plateau at the 1-year follow-up and remained stable to the 3-year follow-up. One year after treatment, approximately 60% of the patients could ambulate outdoors, and the proportion remained steady until the 3-year follow-up. There was no difference in the 1- and 3-year follow-up anti-osteoporosis medication administration rates (approximately 40%).This study confirms the benefits of having a multidisciplinary FLS care team to manage older people with FHF. An FLS improves the care of patients with FHF and the social support of caregivers and relatives. The FLS maintained the functional outcomes of the patients through 3 years of postfracture treatment. Fragility hip fracture (FHF) is one of the most severe complications of osteoporosis . WithoutConcerted efforts to improve the diagnosis, treatment, and prevention of FHFs are necessary to mitigate their ongoing strain on national economies and societies. Several programs have therefore been developed, such as the “Own the Bone”  and “CapFew studies on FLS have been conducted in Thailand. In addition, some core clinical outcomes are underreported, such as postinjury mobility, performance in activities of daily living, and quality of life after using an FLS . TherefoPatients diagnosed with a pathological fracture confirmed by a pathological study. These patients were excluded since their prognoses differ from those of patients with an osteoporotic fracture.Patients who sustained multiple injuries or fractures. This group of patients was excluded because their rehabilitation programs and recovery are different from those of patients sustaining a hip fracture only.Before this research began, the Institutional Review Board approved its protocol (COA no. Si 754/2019). We retrospectively reviewed patient data recorded in the Siriraj–FLS Registry between July 2016 and October 2018. The investigation only assessed patients who had been diagnosed with femoral neck and intertrochanteric fractures and who had a minimum follow-up of 1 year or until death. The exclusion criteria were as follows:A multidisciplinary care team assessed all patients with an FHF and treated them according to our center’s hip fracture protocol . After tmortality raterefracture rate the proportion of patients receiving calcium and vitamin D supplementationthe proportion of patients given anti-osteoporosis medicationsDemographic data and clinical information were collected as follows: age, sex, body mass index, Charlson Comorbidity Index, the percentage of 10-year probability of fracture by the fracture risk assessment tool (FRAX) , historyactivities of daily living (using the Barthel Index)health-related quality of life postfracture ambulatory statusIn addition, we determined the bone mineral density (BMD) assessment rate during the first year after FHF treatment. We also collected details of functional outcome measures as follows:Patients’ postfracture ambulatory statuses were classified as bedridden, indoor ambulator, and outdoor ambulator. All outcome measures were evaluated at 3 time points: before discharge, and 1 and 3 years after treatment. The postdischarge evaluations were conducted by telephone interviews with the patients or, if they could not communicate via telephone, their primary caregivers.The Barthel Index (BI) is a 10-item ordinal scale used to evaluate patients’ functional independence in performing their activities of daily living. Mahoney and Barthel DW introduced the BI in 1965 . It has The EuroQoL-Visual Analogue Scale (EQ-VAS) records a patient’s self-rated health status on a vertical 20-cm visual analog scale. Its grading ranges from “0”  to “100” . Patients mark an “X” on the scale in the position that reflects their perception of their current health status. This tool has been validated in older adults with FHFs , 14.P) values ≤ 0.05 were considered statistically significant.Descriptive statistics were used. Continuous variables are presented as the means, standard deviations, and ranges, while categorical variables are summarized as frequencies and percentages. Changes in BI and EQ-VAS scores from discharge to 1 and 3 years after a hip fracture were assessed with a one-way repeated-measure analysis of variance (one-way repeated ANOVA). The BI and EQ-VAS scores at discharge and the 1- and 3-year follow-ups were compared using post hoc analysis with Bonferroni correction. Data analyses were performed using PASW Statistics for Windows, version 18 . Probability . The average body mass index was approximately 22.3 kg/mDuring the 3-year follow-up, the cumulative deaths were 13, 68, and 100 at discharge and 1 and 3 years after hip fracture treatment, respectively. Thus, the in-hospital mortality rate of our patient population was 2.7%; the rate rose to 13.9% and 20.4% at 1 and 3 years after hip fracture treatment, respectively. During the data collection period, excluding deaths, none of our patients was lost to follow-up. Therefore, the total numbers of patients available for statistical analysis were 476, 421, and 389 at discharge and the 1- and 3-year follow-ups, respectively .During hospitalization, all patients and their caregivers received video-based osteoporosis educational material. BMD assessments were carried out on 397 patients 81.2%) 1 year after their FHFs. The proportion of patients who received calcium and vitamin D supplementation was 98.3% at discharge, and this proportion remained at approximately 90% during the 3-year follow-up. Regarding anti-osteoporosis medications, the rate of prescribing anti-osteoporosis medication before discharge was only 13.4%. At the 1- and 3-year follow-ups, the proportion of patients using anti-osteoporosis medication had risen to approximately 40%  and remained stable (P = 1.000) until the 3-year follow-up .With the populations in many countries around the world aging rapidly, higher incidences of fragility fractures are inevitable. These fractures are associated with increased disability, morbidity, and mortality. If these problems are not satisfactorily addressed, the rise in fragility fractures will adversely impact patients’ health and place substantial economic burdens on societies –18. Our Compared with other reports from the Asia–Pacific region , a substIn addition, the refracture rate of our Siriraj-FLS patients (5.7% at the 3-year follow-up) was considered low and comparable to those reported in a systematic review (0–6.5%)  and a meOur low refracture rate might result from several factors. One is our comprehensive fall prevention program, which is part of the osteoporosis education given to all patients and their caregivers. The other factor is the high rate of BMD testing, which facilitates the prevention of secondary fragility fractures by identifying patients at risk and encouraging their compliance with anti-osteoporosis therapy. It is also important to note that over 90% of our patients received calcium and vitamin D supplementation. As for those who did not receive supplementation, most had serum calcium and vitamin D levels within the normal ranges.P = 1.000 for both EQ-VAS and BI). Our mean post-hip fracture BI and EQ-VAS scores are comparable to the scores reported by previous studies. For instance, Chiang et al. [The mean 1-year BI and EQ-VAS scores increased dramatically from their baseline values. Afterwards, there were no statistically significant differences between our BI and EQ-VAS scores at the 1-year follow-up and their corresponding values at the 3-year follow-up (g et al.  and Imaig et al.  reportedg et al.  and the g et al. Our 1- and 3-year mortality rates were 13.9% and 20.4%, respectively. These numbers were compatible with those reported in a previous systematic review and meta-analysis of various centers with an FLS care model , 19. In Interestingly, in 2005, only 4% of patients in Thailand received anti-osteoporosis medications after hip fracture in centers without an FLS . Our antThe strength of this study is that our FLS collected data related to a range of core outcomes. They were pre- and postinjury ambulation, performance in activities of daily living, and quality of life after FLS implementation. These outcomes have tended to be underreported in the literature, yet they are crucial to determining the comprehensive status of patients with FHFs. Nonetheless, there are some limitations to this study. First, our study had a retrospective design; nevertheless, the fact that we collected data from our FLS registry minimized potential biases. Second, this study drew upon data from only one center in Thailand, a high-volume hospital with an experienced FLS team. Consequently, some aspects of our data and findings may not be generalizable to centers that provide a less sophisticated level of care or do not have an FLS.In conclusion, our findings concur with previous reports that FLSs contribute to significant improvements in the rates of osteoporosis education and calcium and vitamin D supplementation, with resulting satisfactory functional outcomes and excellent BMD and refracture rates. Because FLSs are highly beneficial to patients with fragility fractures, they should be established at all centers. Nevertheless, only approximately 40% of our patients with FHFs adhered to their anti-osteoporosis treatment. Further study is needed to identify the reasons for unsatisfactory anti-osteoporosis compliance, and specific interventions should be explored to increase the rate of anti-osteoporosis initiation.


Background: Vitamin D is considered a potent modulator of the immune system, albeit its role in COVID-19 infection is a matter of debate. The present study aimed to estimate the association between serum vitamin D levels and COVID-19 among people in Qatar.Methods: This case-control study, approved by the Institutional Review Board of Primary Health Care Corporation (PHCC) Qatar, retrospectively evaluated the principal public healthcare sector population data repository retrieved from the cloud-based Electronic Health Record (EHR) software-Cerner, during April 2020–2021. The health records of all adult patients aged >18 years who had undergone the reverse transcription-polymerase chain reaction (RT-PCR) test and whose medical records had documented serum 25-hydroxyvitamin D [25 (OH)D] levels were analyzed.p < 0.001).Results: A total of 924,173 EHRs were extracted, of which 62,451 EHR comprised of 16,446 (26.3%) COVID-19 patients and 46,005 (73.7%) negative-control group patients met the inclusion criteria. The odds ratio (OR) among different categories of vitamin D deficiency (VDD) revealed that people with mild/moderate VDD were 1.18 times (95% CI 1.126–1.258) and those with severe VDD were 1.90 times (95% CI 1.116–1.251) more likely to have COVID-19 infection when compared to the people with optimal serum vitamin D level. On applying multiple logistic regression, the odds of having COVID-19 infection were found to be 1.27 times (95% CI 1.184–1.371) higher among those with mild/moderate VDD and 1.32 times (95% CI 1.206–1.405) higher among those with severe VDD when compared to people with optimal vitamin D level (Conclusion: Our findings demonstrated a significant association between the suboptimal serum vitamin D level and COVID-19 infection. Further studies are required to determine the effects of VDD on the severity and outcomes of COVID-19 infections. This international public health crisis of substantial mortality has accelerated research to elicit its association with various conditions/diseases and to explore avenues for its prevention.Since the time it was first identified in the Chinese city of Wuhan in December 2019 until date, the COVID-19 pandemic continues to present daunting challenges of immense magnitude to human survival, health, and well-being across the world. COVID-19 remains an enigma. Multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and spread across the world, leading to the massive upheaval of fear, anxiety, stress, and depression among the general population and especially among healthcare workers. Vitamin D is considered a potent modulator of the immune system. Therefore, several researchers examined the plausible association between vitamin D deficiency (VDD) and COVID-19 infections. However, the evidence was inconsistent and inconclusive as some studies indicated an association but others did not. This finding may be attributed to variations in methodology, small samples, confounding factors, or diverse healthcare systems. Hence, it is imperative to accumulate evidence and better understand the role of VDD in the COVID-19 pandemic. The prevalence rate of VDD among people in Qatar is exceptionally high, reaching 71.4%. Owing to the extensive skin coverage, scarce exposure to natural sunlight, and longer in-door dwelling to avoid the extreme soaring heat and arid climate most of the year, it is of considerable interest to determine the association of serum vitamin D levels with COVID-19 infection in this country. It is hypothesized that VDD is found frequently among people testing positive for COVID-19 infection. Therefore, this retrospective study was undertaken to estimate the association between serum vitamin D levels and COVID-19 among people who undertook reverse transcription-polymerase chain reaction (RT-PCR) test for COVID-19 infection during the study period of April 2020–2021.Several studies have reported the potential of micronutrients like zinc, selenium, and iron in modulating the immune system and their deficiency correlating with COVID-19 infection.2, located amid the western coast of the Arabian Gulf. The population of Qatar at the end of April 2021 was 2,646,854 people . Qatari nationals account for approximately < 15% of the total population, with the remaining being comprised of expatriates. The first positive COVID-19 patient was reported in Qatar on February 29, 2020.Qatar is a peninsula state, with an area of 11,521 km The HMC operates 12 hospitals  and provides specialist facilities and services for secondary and tertiary care in the country.This case-control study retrospectively evaluated the principal public healthcare sector population data repository of the Primary Health Care Corporation (PHCC) and Hamad Medical Corporation (HMC) retrieved from the cloud-based Electronic Health Record (EHR) software—Cerner. PHCC delivers primary healthcare services through 27 health centers strategically located in 3 main health regions—northern, central, and western, covering the entire State of Qatar.Health records of all adult patients aged >18 years who had undergone RT-PCR test and whose medical records, including the serum 25-hydroxyvitamin D [25 (OH)D] levels, were recorded in the EHR were analyzed . Vitamin D assessment was considered valid if it was conducted within a year before the date of RT-PCR testing. Patients whose serum vitamin D level was assessed within 14 days of the RT-PCR test for COVID-19 were excluded, as this duration coincided with the incubation period for coronavirus. Patients who had received any form of vitamin D supplementation (tablets/injection) after serum 25 (OH) D testing and before the RT-PCR test for COVID-19 were excluded from the study.This study was approved by the Institutional Review Board of Primary Health Care Corporation, State of Qatar (Reference no: PHCC/DCR/2021/05/034). The data analysis was performed without the use of any identifiable data. However, demographic characteristics, serum vitamin D level, COVID-19 infection status, smoking status, body mass index (BMI), and the medical history of comorbidities/medication related to COVID-19 and vitamin D metabolism were extracted by the Business and Health Intelligence team of PHCC.The assessed cases included patients with positive testing on RT-PCR for COVID-19, based on the national guidelines, whereas those with negative RT-PCR test results constituted the control group. Both COVID-19 patients and the control subjects were representatives of the population in Qatar during April 2020–2021 time frame. RT-PCR test was performed using nasopharyngeal and/or oropharyngeal swabs and was considered positive based on the quantitative detection of ribonucleic acid from the SARS-CoV-2 virus at the Department of Laboratory Medicine and Pathology, HMC.The serum vitamin D level assessment reports were retrieved from EHS. The Serum 25 (OH)D levels were categorized as follows: severe VDD (serum 25 (OH)D < 10 ng/mL), mild/moderate VDD category (10–20 ng/mL), and optimal serum vitamin D (>20 ng/mL).Data cleaning involved the elimination of the population with invalid serum vitamin D reports and checking for dates of COVID-19 testing within the specified study time range. Data analysis was performed on SPSS 28 .2, Negelkerke R2, and Hosmer–Lemeshow goodness-of-fit tests.The continuous variables were presented as the means and standard deviations, while frequencies and percentages were used to express the categorical variables. The Chi-Square test was performed to assess the association between categorical variables, and the Mann–Whitney U-test was performed to compare the means between COVID-19 patients and control subjects. The significance level was set at 5%. The logistic regression model accounted for the confounding variables and shortlisted the most significant variables. All variables were initially entered into the model. Subsequently, significant variables were further analyzed in another model, and the adjusted odds ratio (OR) was calculated. Finally, the models were evaluated using the Omnibus test, Cox Snell RA total of 924,173 EHRs were extracted, which comprised the potentially eligible population who had undergone RT-PCR testing for COVID-19 during April 2020–2021. Among these, 62,451 had valid serum 25(OH) D assessment documented in the EHR and were of age >18 years. Hence, the final study sample consisted of 16,446 (26.3%) COVID-19 patients who were RT-PCR-positive and 46,005 (73.7%) control subjects .p = 0.48). When the study population was stratified by age, a statistically significant difference (p < 0.001) was detected between the groups, with more people in the 30–39 and 40–49 year-age groups who tested RT-PCR positive. Age distribution of the COVID-19 patients and the control subjects are depicted in p < 0.001).The general characteristics of the study population are shown in p < 0.001). The mean BMI among COVID-19 patients was 30.24 ± 7.32 kg/m2, which was significantly marginally higher than that of the control subjects (29.64 ± 8.14 kg/m2) (p < 0.001). A greater proportion of people in the COVID-19 group (46.3%) were obese relative to the control group (41.1%) (p < 0.001). Gender distribution and smoking status showed no statistically significant difference between the two groups.A statistically significant variation in COVID-19 patients was also observed by nationality. Lower disease prevalence was detected among Qataris (30.7%) when compared to non-Qataris (69.3%) (p < 0.001).p < 0.001)  higher among those with mild/moderate VDD and 1.32 times (95% CI 1.206–1.405) higher among those with severe VDD when compared to people with optimal vitamin D level (< 0.001) . TestingThis study is one of the few, large-scale, retrospective case-control studies to date to investigate the role of vitamin D in COVID-19 infection. Multivariate analysis of our data suggested a statistically significant association between the suboptimal serum vitamin D level and COVID-19 infection after controlling for the significant confounding variables. Hence, suboptimal serum vitamin D level was independently associated with COVID-19 infection. Another well-recognized role of vitamin D is immunomodulation, which encompasses its critical influence on innate and adaptive immunities. The putative role of vitamin D in the immune response against viral infections such as influenza, dengue, hepatitis, and HIV has been extensively researched. Amid the ongoing COVID-19 pandemic, the contributory influence of VDD has drawn great interest and driven numerous scientific publications to add to the existing body of knowledge.The vital role of vitamin D in the regulation of calcium and phosphorus metabolism within the human body and its deficiency impacting the musculoskeletal system is indisputable. It also promotes the degradation of the SARS-CoV-2 virus through endolysosomes. VDD weakens the innate immune response and enables higher SARS-CoV 2 load, which results in the overactivation of the adaptive immune system with increased release of cytokines such as TNFα and IL-1β, leading to the production of a cytokine storm and progression in the severity of the infection and increased mortality. VDD also causes the overactivation of the pulmonary renin-angiotensin system (RAS), leading to respiratory syndrome. RAS is partly regulated by angiotensin-converting enzyme 2 (ACE2), which also acts as a primary receptor for SARS-CoV-2 entry into the cells. Hence, VDD can exacerbate COVID-19 through its effects on ACE2.It has been ascribed that the contributory role of vitamin D in COVID-19 infection is pleotropic. First, vitamin D supports the production of antimicrobial peptides in the respiratory epithelium by monocytes and neutrophils. concluded that individuals with an insufficient level of vitamin D were 80% more likely to acquire COVID-19 infection when compared to those with a normal level of vitamin D . Another meta-analysis of 23 studies including 11901 patients by Ghasemian et al. suggested the odds of getting infected with SARS-CoV-2 to be 3.3 times higher among individuals with VDD (95% CI: 2.5–4.3) and the chance of developing severe COVID-19 to be approximately 5 times higher in patients with VDD .Previous systematic reviews and meta-analyses have clearly indicated an association between vitamin D levels and COVID-19 infection. Teshome et al. Another study among 156 COVID-19 patients and 204 controls in India revealed a significant association between vitamin D levels and COVID-19 infection with an adjusted OR of 3.295 (1.25–8.685). A study from Wuhan revealed, through multiple logistic regression, that VDD is associated with COVID-19 infection.Similar to our findings, Meltzer et al. conducted multivariate analysis among 489 patients and reported a relative risk of testing positive for COVID-19 to be 1.77 higher among patients with VDD status.Abdollahi et al. from Iran, Ye et al. from China, Hernández et al. from Spain, Carpagnano et al. from Italy, Angelidi et al., from the USA, and Diaz-Curiel et al. from Spain also reported significantly higher rates of VDD in COVID-19 patients. This difference may be attributed to the limitation in the analytical approach applied as they took into account the serum 25 (OH) D levels of 10–14 years before the COVID-19 pandemic and were not contemporaneous.Conversely, a study on 348,598 UK Biobank participants did not find any significant association between VDD and testing positive for COVID-19 infection. who investigated the roles of environmental factors in the spread and transmission of COVID-19 infections. Qu et al. found that respiratory tract infections were more frequent in the winter seasons, particularly in the northern latitudes, when compared to the summer season, with high infections of COVID-19 reported during the winter months. Our study revealed evidence that VDD is a significant contributor to COVID-19 infection, especially in the winter.Our study's strength lies in the investigation of the important contribution of seasonal variations in vitamin D levels on COVID-19 infections. We found that individuals with serum vitamin D levels assessed during winter were more likely to have COVID-19 infection. These findings agree with those of Qu et al.We acknowledge that the results of this study were based on patient data from the principal public healthcare sector of Qatar and may not be nationally representative as the semi-government, private, and voluntary health institutions were not included.Our results revealed a strong and significant association between the suboptimal serum vitamin D level and COVID-19 infection after controlling for the confounders in the regression model. The VDD status, as indicated by low 25(OH)D levels, strongly indicated a contribution to the development or onset of COVID-19 infection. Hence, daily vitamin D supplementation may be recommended as the potential strategy to prevent COVID-19, particularly for vulnerable adults, obese adults, and individuals with comorbidities. Further studies are warranted to determine the effects of VDD on the severity and outcomes of COVID-19 infections.The authors declare no conflicts of interest.All authors contributed to the design, data collection, data analysis, and interpretation of the results. NAA, BNFJ, HAA and SAA led the data collection. ASG led the data analysis. NAA, BNFJ, ASG, SAA and HAA led the interpretation and presentation of results. NAA and BNFJ wrote the initial draft of the manuscript. All the authors read and approved the final manuscript.


The short- and medium-range order of the constructed a-SiCThis paper evaluates the mechanical properties of amorphous silicon carbon nitride (a-SiC Amorphous ternary silicon carbon nitride  magnetron sputtering exhibited dielectric stability and temperature-invariant dielectric tunability at an elevated temperature of 673 K . Throughy alloys . The locy alloys . MoreoveIn this paper, the structural properties and mechanical structural properties of a-SiCThe molecular structure of a-SiCapproach . First, The initial structures are melted at 3000 K to obtain a stable configuration, specifically, equilibrated for 50 ps in the canonical (NVT) ensemble and 150 ps in the isothermal-isobaric (NPT) ensemble. The structure decreases linearly from 3000 K to 300 K at a cooling rate of 2.5 K/ps. Such a cooling process is in an NPT ensemble, allowing the structure to shrink in response to the applied temperature. At a temperature of 300 K, the obtained structure is equilibrated for 40 ps in the NVT ensemble, followed by the NPT ensemble for another 40 ps before calculating properties. The constructed representative atomic structure of a-SiCby OVITO .r, as shown in Equation  of SiCN and g(r) of a-SiCThe RDF quantifies the short-range order, while structure factors characterize the medium-range order.  reduced .The change in the peak height and position of the RDF reveals the structural difference between various a-SiC lengths . In partThe structure is assessed by calculating the total structure factors S(q) for a-SiC37C32N31 . The mecThe stress–strain curves of bulk amorphous SiC (a-SiC), Sius Si3N4 . The ducThe elastic modulus is estimated from the initial slopes of the linear portion of the tensile strain–stress curves. The linear part is from the origin to the point corresponding to a strain of 0.1. The calculated elastic moduli and Poisson’s ratios are shown in f 870 °C , and 240f 870 °C . For a-S–290 GPa , while tAccording to  241 GPa  as the n 120 GPa . The var 120 GPa . The resYoung’s modulus of a-SiCThis paper presented an MD simulation-based evaluation of the structure and mechanical properties of a-SiC


Acute kidney injury (AKI) is a common disease that can develop into end-stage kidney disease. Sepsis is one of the main causes of AKI. Currently, there is no satisfactory way to treat septic AKI. Therefore, we have shown the protective function of Cul4a in septic AKI and its molecular mechanism.The cellular and animal models of septic AKI were established by using lipopolysaccharide (LPS). Western blot (WB) was employed to analyze Cul4a expression. RT-qPCR was employed to test the expression of Cul4a, SOD1, SOD2, GPX1, CAT, IL-6, TNF-a, Bcl-2, IL1b, Bax and KIM-1 mRNA. ELISA was performed to detect the contents of inflammatory factors and LDH. CCK-8 was utilized to detect cell viability. Flow cytometry was utilized to analyze the apoptosis. DHE-ROS kit was used to detect the content of ROS.Cul4a was down-regulated in cellular and animal models of septic AKI. Oxidative stress is obviously induced by LPS, as well as apoptosis and inflammation. However, these can be significantly inhibited by up-regulating Cul4a. Moreover, LPS induced the activation of the NF-kB pathway, which could also be inhibited by overexpression of Cul4a.Cul4awas found to be a protective factor in septic AKI, which could inhibit LPS-induced oxidative stress, apoptosis and inflammation of HK-2 cells by inhibiting the NF-kB pathway. AKI refers to a syndrome in which a patient's renal function is significantly declined rapidly due to various causes, and a series of clinical symptoms occur, including increased serum creatinine (Cr), decreased urine output, electrolyte disturbance, acidbase imbalance, etc. In severe cases, acute brain edema, acute heart failure, or even life-threatening may occur. Suffering from AKI can lead to increased demand for renal replacement therapy, increased risk of death, and more treatment costs, which will bring a heavy burden to patients and society Sepsis is a continuous and excessive inflammatory response and immunosuppression caused by pathogen invasion. It is an important factor for organ failure of the body and can lead to shock and even death of patients Cullin4A (Cul4a) belongs to the E3 ligase ubiquitin family in the ubiquitin-proteasome system (UPS) and determines the substrate specificity of ubiquitination modification Here, we describe a new role of Cul4a, which is to inhibit inflammation and apoptosis mediated by oxidative stress in septic AKI. This will provide a potential new treatment for septic AKI.Ten male Sprague-Dawley (SD) rats  were raised in an SPF environment. The breeding room has a temperature of 22-25°C and a humidity of about 50%. LPS (5 mg/kg) was injected intraperitoneally to establish a septic AKI model.2. The medium needs to be changed every 24 hours. 500 ng/mL LPS was used to induce cell damage.HK-2 cells, human renal cortex proximal convoluted tubule epithelial cell line, were purchased from Yaji Biotechnology Co., Ltd . The cells were cultured in a culture medium composed of DMEM/F-12  and 10% FBS  at 37°C with 5% COThe Cul4a overexpression plasmids  was transfected into HK-2cells using Lipofectamine™ 3000 in accordance withthe instructions.Radioimmunoprecipitation assay lysis buffer  was used to extract the total protein in HK-2 cells. The concentration was examined by the BCA method. After incubation with the loading buffer, the same amount of protein (30 mg) from each group was added into SDS-PAGE. The voltage was set to 120 volts. When the protein is sufficiently separated, it is transferred to the PVDF membrane. The current was set to 300 mA. After the membranes were blocked by QuickBlock™ Blocking Buffer , primary antibodies  were added and incubated at 4°C. The next day, the secondary antibody was used to incubate the membranes. The electrochemiluminescence (ECL) developer was added dropwise to develop imaging, and the grey value was semi-quantitatively analyzed according to Image J software.The TRIzol reagent  was used to extract total RNA in HK-2 cells following the protocols. The complementary deoxyribose nucleic acid (cDNA)was synthesized using Real Master Mix . RT-qPCR was performed using the Prism 7900 System. GAPDH was utilized to normalize the expression of mRNAs. All the primers were listed in The level of MDA in the supernatant of HK-2 cells was examined by Lipid Peroxidation (MDA) Assay Kit  according to the instructions.The contents of ROS in HK-2 cells was tested using DCFH-DA . The cells were incubated with DCFH-DA (5 mmol/L) for 30 min in the dark. Then the cells were collected by 0.05% trypsin-EDTA solution. After that, the cells were suspended in a fresh medium. Finally, the level of ROS was tested by a flow cytometer.The supernatant of HK-2 cells was collected. The contents of inflammatory cytokines  and LDH were detected by commercial ELISA kits  following the protocols.The HK-2 cells were collected by trypsin and centrifugation. Then the cells were resuspended in a 200 mL binding buffer. After that, 5 mL Annexin V-FITc and 5 mL PI were added into the binding buffer. Finally, 10 min later, the apoptosis rate was measured by a flow cytometer.The apoptosis was examined by TUNEL staining with a TUNEL kit  following the manufacturers' instructions. The nucleus was stained by DAPI. The images were observed by the inverted fluorescence microscope.The measurement data were described as the mean±standard deviation (SD). All statistical analyses were performed by GraphPad Prism 8.0. One-way analysis of variance (ANOVA) or Student's t-test was used for comparison. Significance was accepted at P < 0.05.First, the expression of Cul4a in the LPS-treated HK-2 cells was detected through WB. Compared with the control group, Cul4a expression in HK-2 cells of the LPS group was significantly reduced A. At theThrough RT-qPCR, we detected the levels of SOD1, SOD2, GPX1, and CAT mRNA. Compared with the control group, the levels of those mRNA in the LPS group were remarkably reduced, suggesting that oxidative stress exists in septic AKI. However, compared with the LPS+vector group, the levels of those mRNA in the LPS+Cul4a group were significantly increased  D. The coWe also tested the inflammatory response in HK-2 cells. LPS obviously induced the production of inflammatory cytokines  mRNA in HK-2 cells. While up-regulating Cul4a suppressed their expression C. We alsThrough the CCK-8 assay, the cell viability was detected. The treatment of LPS significantly reduced the viability of HK-2 cells, but overexpression of Cul4a could reverse this A. FurtheSince the NF-kB pathway plays an important role in oxidative stress, inflammation, and apoptosis, we tested the marker proteins of this signalling pathway. The treatment of LPS significantly increased the expression of IκKα but decreased the expression of IκBα. However, overexpression of Cul4a significantly reversed their expression A. The exIn this present study, we revealed the protective role of Cul4a in septic AKI. We have revealed for the first time that Cul4a is down-regulated in septic AKI. Overexpression of Cul4a can significantly inhibit oxidative stress, inflammation and apoptosis, thereby reducing septic AKI. This protective effect was achieved at least in part by inhibiting the NF-κB signalling pathway.The pathogenesis of AKI caused by sepsis is very complicated, which may be related to the increase of inflammatory factors, oxidative stress, and apoptosis NF-κB is a nuclear transcription factor involved in inflammation, oxidative stress and apoptosis Our study demonstrates the regulatory role of Cul4a in septic AKI. Overexpression of Cul4a can remarkably inhibit LPS-induced oxidative stress and inhibit the production of inflammatory factors and apoptosis of HK-2 cells. The protective effect of Cul4a is at least partially achieved by inhibiting the NF-κB pathway.To sum up, Cul4a was found to be a protective factor in septic AKI, and overexpression of Cul4a could inhibit LPS-induced oxidative stress, inflammation, and apoptosis of HK-2 cells by inhibiting the NF-κB pathway.The authors declare that they have no conflict ofinterest.


Vss) of DA-6886 was relatively large (4.91–7.84 L/kg), which was consistent with its high distribution to the liver, kidney, lung, and digestive tract, and was dose-independent. After oral administration, the extent of absolute bioavailability (F) tended to increase (18.9–55.0%) with an increasing dose. The slope of the log-transformed AUC and/or Cmax values versus log dose was greater than unity and greater for oral administration (~1.9) than for intravenous administration (~1.1). Because the nonlinear pharmacokinetics of DA-6886 was more obviously observed after oral administration, it appears that the saturation of pre-systemic intestinal and/or hepatic first-pass extraction of DA-6886 at high doses occurred. DA-6886 is a novel serotonin (5-hydroxytrypamine [5-HT]) receptor 4 agonist for the potential treatment of constipation-predominant irritable bowel syndrome. The purpose of this study was to validate the quantitative assay of DA-6886 in rat plasma and to evaluate the pharmacokinetics and tissue distribution of DA-6886 in rats. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the robust quantification of DA-6886 in rat plasma was successfully validated and applied to the pharmacokinetic studies in rats. The pharmacokinetic parameters of DA-6886 in rats were evaluated following single intravenous or oral administration at three dose levels . DA-6886 exhibited a smaller dose-normalized area under the plasma concentration–time curve (AUC) values and faster clearances in the low-dose group than in the high-dose group following both intravenous and oral administration. The steady-state volume of distribution ( In addieceptors . The aff agonist . The pha agonist .The aim of this study was to validate the quantitative analysis method of DA-6886 in rat plasma and to evaluate the pharmacokinetics and tissue distribution of DA-6886 in rats. The pharmacokinetics of DA-6886 and its dose-dependency are important data for the design of clinical studies and a comprehensive understanding of safety and efficacy of the drug during preclinical development. In the present study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the precise and accurate quantification of DA-6886 in rat plasma. The dose-dependency in the pharmacokinetics of DA-6886 was verified following a single intravenous and oral administration of DA-6886 at various doses to rats. The distribution of DA-6886 to various tissues was also studied following its single intravenous administration.DA-6886  was a product of Dong-A ST . Verapamil hydrochloride  and acetic acid (cat no. 40512) were purchased from Sigma-Aldrich . Other reagents and solvents were of reagent or HPLC grade.The protocols of animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Dong-A Research Center  and conducted according to the IACUC guidelines. Male Sprague-Dawley rats purchased from Orient Bio  were acclimated for 1 week and used for studies at ~8 weeks old. The conditions and procedures used for the housing and handling of rats are similar to those described in other studies ,13. The n = 4 each). Blood samples (~250 μL) were collected via the carotid artery at 0, 1 (only for intravenous administration), 5, 15, 30, 60, 120, 180, 300, 420, and 1440 min following the drug administration. The cannula was flushed following each blood sampling to avoid blood clotting using 0.4 mL heparinized (20 units/mL) normal saline. The collected sample was immediately centrifuged and a 100 μL plasma sample was stored at −20 °C until it was used for the LC–MS/MS analyses of DA-6886. The jugular vein (for intravenous administration only) and carotid artery  were cannulated with polyethylene tubing (PE 60) according to reported methods ,13. DA-6n = 5 for each time point). Each organ was homogenized by adding five times the volume of sterile distilled water. After centrifugation at 4 °C for 3 min, 20 μL of each supernatant was dispensed and stored frozen at −20 °C.After the intravenous administration of 10 mg/kg DA-6886 in the same way as above, at 0.5 and 5 h, plasma and major organs  were enucleated (v/v)) was accomplished with flow rate of 0.2 mL/min. The column and autosampler temperatures were set at 30 °C and 4 °C, respectively. The total run time was 5 min. The LC–MS/MS system consisted of a Shiseido Nanospace SI-2 LC System  and an API 4000 QTrap mass spectrometer . The instrument was controlled using the Analyst software . The LC–MS/MS method for the determination of DA-6886 concentrations in the samples was developed and validated for its linearity, sensitivity, specificity, precision, accuracy, matrix effect, recovery, stability, and dilution effect . Chromatm/z 407.195 → 184.000 for DA-6886 ([M+H]+) and m/z 455.200 → 165.000 for the IS ([M+H]+).The eluent was monitored using a multiple reaction monitoring mode of mass spectrometer equipped with an ESI source in positive ion mode. The instrument parameters were as follows: nebulizer gas (50); curtain gas (20); auxiliary gas (50); collision gas (medium); ion spray voltage (5.5 kV); entrance potential (10); and temperature (500 °C). The declustering potential, collision energy, and collision cell exit potential were set at 86, 41, and 10 V for DA-6886 and 101, 37, and 8 V for the IS (verapamil). The precursor-to-product ion transitions applied for quantification were A 300 μL aliquot of IS working solution in acetonitrile (verapamil 50 ng/mL) was added to each 100 μL plasma sample and mixed by vortex to precipitate the protein. A 500 μL of the IS working solution was added to the tissue samples dispensed by 20 μL, respectively. The preparation of calibration standards for tissue samples is detailed in Vss), were calculated using non-compartmental analyses  [0–∞) [Cmax) and time to reach Cmax (Tmax) were determined from the observed data. The extent of absolute oral bioavailability (F) was estimated by comparing the AUC0–∞ of DA-6886 after oral administration with the AUC0–∞ of DA-6886 after the intravenous administration of the same dose. All data are presented as mean ± standard deviation (S.D.), except median (range) for Tmax. An assessment of the dose proportionality was performed using the reported power model based on 90% confidence interval (CI) for the ratio of dose normalized means (Rdnm) lying within pre-specified acceptance limits (0.8–1.25) [Pharmacokinetic parameters, such as time-averaged total body clearance (CL) and steady-state volume of distribution  . The traA) [0–∞) . The max.8–1.25) ,18.p value of <0.05 was obtained. GraphPad Prism  was utilized for statistical analysis. To compare three means of unpaired data, the Kruskal–Wallis test and a post hoc Dunn’s multiple comparison test were applied. Statistical significance was concluded when a The calibration curve was constructed by calculating the peak area ratio of DA-6886 to the peak area of verapamil (IS), and it showed a linearity of r = 0.9980 to 0.9997 in the concentration range of 2 to 2000 ng/mL. The relative error (RE) for the concentration of each standard sample, which was inversely converted using the calibration curve of DA-6886, was −3.14 to 3.04%, and the coefficient of variation (CV) was 1.73 to 5.21%. The lowest limit of quantitation (LLOQ) of this assay was that the peak area of DA-6886 was at least five times higher than that of the empty plasma sample, and the concentration was determined to be less than 20% error in precision and accuracy. The LLOQ of this assay was determined to be 2 ng/mL , and at n = 5) for each concentration sample was 2.16–5.00% (n = 5) was 2.45~4.12% (n = 5) for each concentration sample was −3.25 to 2.58% (n = 5) was 0.733~3.33% (The precision of the assay is determined by the CV for the concentration of DA-6886 measured using QC samples at low (6 ng/mL), medium (100 ng/mL), and high (1600 ng/mL) concentrations. The CV value for intra-day precision  and high concentration QC (HQC) samples. The CV of the peak area for each concentration were 5.83% (at 6 ng/mL) and 3.06% (at 1600 ng/mL), which satisfies 15% or less. The recovery of DA-6886 during the sample preparation was estimated by comparing the peak area of DA-6886 in the LQC and HQC samples (extracted samples) to that of the unextracted samples prepared by adding the working standard of DA-6886 equivalent to the LQC (6 ng/mL) and HQC (1600 ng/mL) after the pretreatment of the empty plasma. The recovery of DA-6886 was 102% and 88.1% in LQC and HQC, respectively, and the CV value was 1.69~6.34%, which satisfies the criteria of less than 15% .n = 3) with a concentration of DA-6886 of 40,000 ng/mL were prepared and measured by pre-treatment after diluting them 20-fold with empty plasma. The CV value for the measured concentration of DA-6886 diluted 20 times was 4.13%, and the RE value was −4.00%. The mean of the measured values for LQC (6 ng/mL) and HQC (1600 ng/mL) following storage with various conditions  were compared with the mean measured values of the immediately prepared QC sample . The CV 0–∞ in the low dose group.The above-validated LC–MS/MS method was applied to the pharmacokinetic studies of DA-6886 in rats. After a single intravenous administration of various doses of DA-6886  to rats, the mean plasma concentrations of DA-6886 versus time are plotted in 0–∞ in the low dose group was significantly smaller than in the high dose group. Greater dose-normalized AUC0–∞ and Cmax were observed with an increasing oral dose of DA-6886. As a result, the F value showed a tendency to increase with an increasing dose. Following a single oral administration of DA-6886  to rats, the mean plasma concentration of DA-6886 versus time profiles are presented in Cmax values versus log dose and their 90% CI were estimated based on the power model  to rats, the slope of the log-transformed AUC and/or er model . The ratAfter a single intravenous administration of 10 mg/kg DA-6886, the concentration and tissue-to-plasma concentration ratio (T/P ratio) in each tissue after 0.5 h and 5 h are shown in 4 receptor agonist for the treatment of IBS, in rat plasma showed acceptable performances in specificity, linearity, sensitivity, precision, and accuracy of analysis. DA-6886 demonstrated robust recovery and stability in rat plasma during storage, sample preparation, and analysis. The successfully validated method was applied to evaluate the pharmacokinetics of DA-6886 in rats at various dose levels. The LC–MS/MS method developed for the quantification of DA-6886, a novel 5-HTdnm of AUC did not lie within the acceptance interval  [Vss of DA-6686 showed similar values irrespective of the intravenous dose. The Vss of DA-6886 was approximately 10-fold larger than the reported total body water in rats (0.668 L/kg) [4 receptor agonist, also showed high distribution in the digestive tract in rats following oral administration [Linear pharmacokinetics with dose-proportional exposure allows us to easily assess dose–effect and dose–toxicity relationships. On the other hand, nonlinear pharmacokinetics may make it difficult to predict effective doses in clinical trials. Therefore, when nonlinear pharmacokinetic parameters are observed in preclinical studies, it is important to elucidate the underlying mechanisms and to quantitively predict nonlinearities in humans ,20. The 8, 1.25) . Based o8, 1.25) , the AUC68 L/kg) , suggeststration . On the Cmax values versus the log oral dose  were much greater than one and the respective slope of intravenous AUC (1.066). Moreover, the 90% CI of Rdnm of oral AUC values lay completely outside of the acceptance interval . Therefore, when DA-6886 was administered orally, the non-proportional increase in AUC according to the dose increase was more pronounced than when administered intravenously. As a result, the F value also increased with an increasing oral dose (18.9% to 55.0%), which is considered to be due to the saturation of the intestinal and/or hepatic first-pass extraction during the absorption process in high dose. This nonlinear pharmacokinetics of DA-6886 observed in rats suggests attention is warranted when carrying out dose escalation during phase I clinical trials. It should be noted that the dose-dependency observed in preclinical pharmacokinetic studies may not necessarily be to the same extent in humans. For example, a drug with different metabolic pathways between species, such as the metabolism in rats with a low Michaelis constant and the metabolism in humans with a high capacity, exhibited nonlinear pharmacokinetics in rats and linear pharmacokinetics in humans [The dose-normalized AUC of DA-6886 also increased with an increasing oral dose. The slope of the log-transformed AUC and n humans . Based o4 receptors are distributed in enterocytes as well as enteric neurons and smooth muscles [In addition to the saturation of the first-pass metabolism of DA-6886, the nonlinear (saturable) distribution of DA-6886 into the intestine during the absorption process could be attributed in part to the dose-dependent pharmacokinetics of DA-6886 after oral administration. Unlike other 5-HT receptors, 5-HT muscles . TherefoF value with an increasing oral dose. The saturation of processes during absorption, such as first-pass metabolism and/or distribution to the intestinal tissue, could be the possible cause of dose-dependency of DA-6886 after oral administration. To quantitatively predict the nonlinearity of the pharmacokinetics of DA-6886 in humans, further studies on the enzyme kinetics of the hepatic and intestinal metabolism in rats and humans are needed. The validated quantification method and the results of this pharmacokinetic study in rats can be utilized for further pharmacokinetic studies of DA-6886. The tissue distribution results can also be applied to the future development of physiologically based pharmacokinetic models of DA-6886.In conclusion, DA-6886 demonstrated nonlinear pharmacokinetics after oral administration (2–20 mg/kg) and showed a larger